Development of a TaqMan-Probe-Based Multiplex Real-Time PCR for the Simultaneous Detection of African Swine Fever Virus, Porcine Circovirus 2, and Pseudorabies Virus in East China from 2020 to 2022

Abstract

African swine fever virus (ASFV), porcine circovirus 2 (PCV2), and pseudorabies virus (PRV) are important DNA viruses that cause reproductive disorders in sows, which result in huge losses in pig husbandry, especially in China. The multiplex qPCR assay could be utilized as a simultaneous diagnostic tool for field-based surveillance and the control of ASFV, PCV2, and PRV. Based on the conserved regions on the p72 gene of ASFV, the Cap gene of PCV2, the gE gene of PRV, and the porcine endogenous β-Actin gene, the appropriate primers and probes for a multiplex TaqMan real-time PCR test effective at concurrently detecting three DNA viruses were developed. The approach demonstrated high specificity and no cross-reactivity with major pathogens related to swine reproductive diseases. In addition, its sensitivity was great, with a detection limit of 10¹ copies/L of each pathogen, and its repeatability was excellent, with intra- and inter-group variability coefficients of <2%. Applying this assay to detect 383 field specimens collected from 2020 to 2022, the survey data displayed that the ASFV, PCV2, and PRV single infection rates were 22.45%, 28.46%, and 2.87%, respectively. The mixed infection rates of ASFV + PCV2, ASFV + PRV, PCV2 + PRV, and ASFV + PCV2 + PRV were 5.22%, 0.26%, 1.83%, and 0.26%, respectively. Overall, the assay established in this study provides an effective tool for quickly distinguishing the viruses causing sow reproductive disorders, suggesting its huge clinical application value in the diagnosis of swine diseases.

Keywords: African swine fever virus; detection; mixed infection; multiplex real-time PCR; porcine circovirus 2; pseudorabies virus.

Figure 1

Standard curves of the multiplex real-time qPCR. Establishment of a standard curve for TaqMan probe-based multiplex real-time qPCR. The logarithm of the number of copies is the horizontal axis. The vertical axis represents Ct values. Each point represents the mean value of three duplicates of plasmid that have been diluted. The optimal standard formula for ASFV is y = 3.242x + 36.79, with a correlation coefficient of 0.9956; the optimal standard formula for PCV2 is y = 3.365x + 38.52, with a correlation coefficient of 0.9965; and the optimal standard formula for PRV is y = 3.357x + 38.64, with a correlation coefficient of 0.9904.

Figure 2

The amplification curves of specificity tests of multiplex real-time qPCR. Only ASFV, PCV2, and PRV exhibited positive fluorescence signals, while other swine pathogens exhibited no fluorescence signals.

Figure 3

The amplification curves of the sensitivity test of the multiplex real-time qPCR. Multiple real-time qPCR sensitivity test using 10-fold serial dilutions of recombinant plasmids, with three repetitions of each dilution test. (A): ASFV sensitivity analysis; (B): PCV2 sensitivity analysis; (C): PRV sensitivity analysis.